Enamel fluorosis is a defect in enamel development that occurs after exposure to excess fluoride. Fluorotic enamel is more porous, and contains more proteins than sound enamel. The mechanisms by which fluoride alters enamel formation remain poorly understood. In this competing renewal, we propose studies to further explore the indirect effects of fluoride resulting from changes in the forming enamel matrix secondary to fluoride incorporation in the growth of enamel crystals. We will also continue to explore the direct effects of fluoride on ameloblast modulation, expression of matrix proteins, proteinases, and ion exchangers that regulate enamel formation. We hypothesize that fluoride-induced amelogenin retention in the early forming matrix, alters pH in enamel compartments, affecting ameloblast modulation and enamel maturation. In late maturation these pH-related effects are further amplified as matrix proteins are removed. This hypothesis will be tested in the following specific aims: 1) To determine the role of fluoride incorporation into the growing enamel mineral on apatite/ protein interactions and processing. 2) To determine how fluoride alters ameloblast modulation and mineral deposition in the maturation stage of enamel formation. 3) To determine the effects of fluoride on the expression of genes related to enamel matrix protein deposition and pH regulation in ameloblasts. We will characterize the effects of fluoride on amelogenin hydrolysis by matrix proteinases both in vitro and in vivo using mass spectrometry. We will then use transgenic mouse models to determine the role of amelogenins and pH ion exchange modulators in buffering the effects of fluoride during enamel development, and finally we will use laser capture microdissection to compare gene expression in ameloblasts at specific stages enamel formation in control and fluoride treated mice. These studies are important to understand how fluoride effects enamel formation, resulting in the formation of fluorosis. By understanding the mechanisms by which fluorosed enamel forms, we will be able to better optimize our use of fluoride to promote dental health.